Methods for obtaining fluid and cellular material from a breast duct

ABSTRACT

Methods for performing medical procedures within the duct of a breast are described. An exemplary embodiment of the invention includes the steps of introducing a guiding member into a breast duct; introducing and positioning a member having an internal lumen in the breast duct; introducing a wash fluid into the breast duct through the internal lumen; removing washings from the breast duct through the internal lumen; and collecting the washings for cytological analysis.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part application and claimspriority under 37 CFR 120 to U.S. application Ser. No. 09/153,564 filedSep. 15, 1998, which is a continuation-in-part application of U.S.application Ser. No. 08/931,786, filed Sep. 16, 1997, now U.S. Pat. No.6,168,779; and this application is a continuation-in-part and claimspriority under 37 CFR 120 to U.S. application Ser. No. 09/740,561 filedDec. 19, 2000 which is a continuation application of U.S. applicationSer. No. 09/067,661, filed Apr. 28, 1998, now U.S. Pat. No. 6,221,622,the contents of each of which are incorporated herein by reference.

[0002] The invention disclosed in this application was made withgovernment support under U.S. Army Medical Research Grant Nos.DAMD17-94-J-4281 and DAMD17-96-C-6117. The government may have certainrights in the invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to medical methods anddevices for accessing body lumens and in particular to methods andapparatus for identifying ductal orifices in human breasts and accessingthe ducts through the identified orifices.

[0005] Breast cancer is the most common cancer in women, with well over100,000 new cases being diagnosed each year. Even greater numbers ofwomen, however, have symptoms associated with breast diseases, bothbenign and malignant, and must undergo further diagnosis and evaluationin order to determine whether breast cancer exists. To that end, avariety of diagnostic techniques have been developed, the most common ofwhich are surgical techniques including core biopsy and excisionalbiopsy. Recently, fine needle aspiration (FNA) cytology has beendeveloped which is less invasive than the surgical techniques, but whichis not always a substitute for surgical biopsy.

[0006] A variety of other diagnostic techniques have been proposed forresearch purposes. Of particular interest to the present invention,fluids from the breast ducts have been externally collected, analyzed,and correlated to some extent with the risk of breast cancer. Such fluidcollection, however, is generally taken from the surface of the nippleand represents the entire ductal structure. Information on the conditionof an individual duct is generally not provided. Information onindividual ducts can be obtained through cannulation and endoscopicexamination, but such examinations have been primarily in women withnipple discharge or for research purposes and have generally notexamined each individual duct in the breast.

[0007] Since breast cancer usually arises from a single ductal systemand exists in a precancerous state for a number of years, endoscopy inand fluid collection from individual breast ducts holds great diagnosticpromise for the identification of intermediate markers. Much of thepromise, however, cannot be realized until access to each and every ductin a patient's breast can be assured. Presently, ductal access may beobtained by a magnification of the nipple and identification of ductalorifice(s) using conventional medical magnifiers, such as magnificationloupes. While such magnified examination is relatively simple, it cannotbe relied on to identify all orifices. Moreover, the ductal orifices canbe confused with other tissue structures, such as sebaceous glands andsimple keratin-filled caruncles of the nipple. Thus, before ductaltechniques can be further developed for diagnostic, research, or otherpurposes, it will be useful to provide methods and apparatus whichfacilitate identification of ductal orifices to distinguish them fromother orifices, and allow subsequent ductal access in selected and/orall ducts in each breast.

[0008] 2. Description of the Background Art

[0009] Publications by the inventors herein relating to breast ductaccess include Love and Barsky (1996) Lancet 348: 997-999; Love (1992)“Breast duct endoscopy: a pilot study of a potential technique forevaluating intraductal disease,” presented at 15th Annual San AntonioBreast Cancer Symposium, San Antonio, Tex., Abstract 197; Barsky andLove (1996) “Pathological analysis of breast duct endoscopedmastectomies,” Laboratory Investigation, Modern Pathology, Abstract 67.A description of the inventors' breast duct access work was presented inLewis (1997) Biophotonics International, pages 27-28, May/June 1997.

[0010] Nipple aspiration and/or the introduction of contrast medium intobreast ducts prior to imaging are described in Sartorius (1995) BreastCancer Res. Treat. 35: 255-266; Sartorius et al. (1977) “Contrastductography for the recognition and localization of benign and malignantbreast lesions: An improved technique,” in: Logan (ed.), BreastCarcinoma, New York, Wiley, pp. 281-300; Petrakis (1993) Cancer Epidem.Biomarker Prev. 2: 3-10; Petrakis (1993) Epidem. Rev. 15: 188-195;Petrakis (1986) Breast Cancer Res. Treat. 8: 7-19; Wrensch et al. (1992)Am. J. Epidem. 135: 130-141; Wrensch et al. (1990) Breast Cancer Res.Treat. 15: 39-51; and Wrensch et al. (1989) Cancer Res. 49: 2168-2174.The presence of abnormal biomarkers in fine needle breast aspirates isdescribed in Fabian et al. (1993) Proc. Ann. Meet. Am. Assoc. CancerRes. 34: A1556. The use of a rigid 1.2 mm ductoscope to identifyintraductal papillomas in women with nipple discharge is described inMakita et al. (1991) Breast Cancer Res. Treat. 18: 179-188. The use of a0.4 mm flexible scope to investigate nipple discharge is described inOkazaki et al. (1991) Jpn. J. Clin. Oncol. 21: 188-193. The detection ofCEA in fluids obtained by a nipple blot is described in Imayama et al.(1996) Cancer 78: 1229-1234. Delivery of epithelium-destroying agents tobreasts by ductal cannulation is described in WO 97/05898.

SUMMARY OF THE INVENTION

[0011] The present invention provides improved methods, kits, and otherapparatus for locating breast ducts in the breasts of human femalepatients. In particular, the methods of the present invention permitreliable identification of the orifices within the nipple of a breastwhich lead to each of the multiple ductal networks within the breast. Byreliably identifying each orifice, all of the ductal networks can belocated and subsequently accessed for diagnostic, risk assessment,therapeutic, research, or other purposes.

[0012] In a first aspect of the present invention, a method for locatingan orifice of a breast duct comprises labelling ductal cells disposed atthe ductal orifice with a visible or otherwise detectable label. Theorifice may then be located based on the presence of the label at theorifice. Specific and preferred methods for labeling the orifices aredescribed below in connection with a second aspect of the presentinvention. After the orifices have been located, an access device, suchas a catheter or fiberoptic viewing scope, can be introduced through atleast one of the orifices and into the associated breast duct. Themethod may further comprise introducing the same or a different accessdevice through other orifices, often into each of the orifices to permitdiagnosis, treatment, or other evaluation of all of the ductal networksof a breast.

[0013] In a second aspect, the present invention comprises a method forlabelling the orifice of a breast duct. The method includes treating anipple to expose tissue in an orifice of each duct. The treated nippleis then exposed to a labelling reagent capable of specifically bindingto a tissue marker characteristic of tissue at the ductal orifice.Binding of the labelling reagent to the tissue results in immobilizationof a label at the orifice, permitting subsequent location of the orificeas described above. The treating step preferably comprises washing thenipple with a keratinolytic agent, such as 5% to 50% acetic acid (byweight), to remove keratin-containing materials which normally occludethe duct orifice and which could inhibit binding of the labellingreagent to the tissue marker. The tissue marker is typicallycharacteristic of the ductal epithelium and represents either a membraneantigen or a cytoplasmic antigen. It has been found by the inventorsherein that the ductal epithelium extends to within 0.1 mm to 0.2 mm ofthe nipple orifice and is sufficiently exposed to the surface of thenipple to permit labelling according to the methods of the presentinvention. Exemplary markers include cytokeratins, such as cytokeratin8, cytokeratin 18, E cadherin, epithelial membrane antigen (EMA), andthe like. Usually, the labelling reagent comprises a polyclonal ormonoclonal antibody or other specific binding substance specific for themarker. The antibody may be directly labelled with a visible label, suchas a fluorescent label, a dye label, a chemiluminescent label, or thelike. Alternatively, the labeling reagent may comprise two or morecomponents, typically including a primary antibody which is specific forthe marker and one or more secondary binding substances which bind tothe primary antibody and provide a label, optionally a magnified label.For example, the primary antibody may be unlabelled, and a secondarylabelled antibody specific for the primary antibody also be provided. Asa further alternative, the primary antibody can be labelled with biotinor other hapten, and binding of the label provided via avidin, secondaryantibody specific for the hapten, or the like. Numerous specifictechniques for labelling of antigenic tissue markers are well known andreported in the immunocytochemical staining literature.

[0014] In a third aspect, a method according to the present inventioncomprises labelling cellular material at a ductal orifice with a visiblelabel and subsequently accessing the duct through the labelled orifice.The labelling step usually comprises the method set forth above. Theaccessing step may comprise introducing an access device through atleast one of the labelled orifices, and preferably through all of thelabelled orifices, where the access device may be a catheter, a fiberoptic viewing scope, or the like.

[0015] In a fourth aspect of the present invention, a kit for labellingbreast duct orifices comprises a labelling reagent or reagents capableof specifically labelling a cellular marker at the ductal orifice,instructions setting forth a labelling method as described above, and apackage containing the labelling reagent and the instructions for use.Usually, the kits of the present invention will further include thekeratinolytic agent and any other reagents that may be necessary forperforming the method. Instructions for use will set forth the use ofall provided reagents and may further set forth the use of agents whichare available in the laboratory where the assay is to be performed.

[0016] In a fifth aspect of the present invention, a kit for accessing abreast duct comprises a labelling reagent capable of specificallylabelling a ductal orifice and optionally a keratinolytic agent fortreating the nipple prior to exposure of the labelling reagent. The kitfurther comprises an access device capable of being inserted through alabelled ductal orifice to a ductal lumen, such as a catheter, a fiberoptic viewing scope, or the like. The kit still further comprises apackage containing the labelling reagent, optionally the keratinolyticagent, and the access device. The accessing kit may further compriseinstructions for use setting forth a method comprising the accessingsteps as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is an anterior view of a human female breast, shown insection, and illustrating three of the six to nine ductal networksextending inwardly from the nipple.

[0018]FIG. 2 is an enlarged view of the nipple of FIG. 1 illustratingthe orifices leading to each of the three ductal networks.

[0019]FIG. 3 is a still further enlarged view of a single orificeillustrating the distribution of tissue markers from the epithelium tothe opening of the orifice, where such markers at the opening areavailable for binding to labelled antibodies.

[0020]FIG. 4 is a schematic illustration of the appearance of a nipplewhich has been labelled with visible markers according to the methods ofthe present invention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0021] The present invention comprises methods for locating, labelling,and accessing the ductal networks in human female breasts. A typicalbreast B is illustrated in FIG. 1 and includes a nipple N and from sixto nine ducts D.

[0022] Three ductal networks D₁₋₃ extending inwardly from the nipple Ninto the breast tissue are illustrated. As best seen in FIG. 2, eachductal network D₁₋₃ begins with an orifice O₁₋₃ which lies at thesurface of the nipple N and extends inwardly through a ductal sinus S₁₋₃and then into a branching network. Each network D comprises a series ofsuccessively smaller lumens which are arranged in complex,three-dimensional patterns. The networks of each duct will overlapwithin the breast tissue but will not be interconnected. The presentinvention relies on identifying and labelling tissue in the orifice O ofeach duct D within the nipple N. Usually, there will be from six to nineorifices which open into a like number of ductal networks. An abrupttransition from the ductal epithelium to the squamous epithelium of theskin is found within about 0.1 mm to 0.2 mm of the nipple surface.Typically, the ductal orifice will be occluded with a conical keratinplug measuring about 0.5 mm to 1 mm in size.

[0023] The present invention relies on the specific labelling of tissuemarkers at the orifice of selected one(s) or all of the ductalnetwork(s). By “specific,” it is meant that the label will be introducedin a manner such that it will bind to the orifice region within thenipple but not bind (or will bind to a significantly lesser extent,usually at least 10-fold less) to other regions of the nipple. In thisway, binding of the label to the orifice will be a discernableindication that the orifice is present and available for access to theassociated ductal network.

[0024] In a particular aspect of the present invention, the tissuemarker(s) will be an antigenic or epitopic site characteristic of theepithelial lining of the breast duct. Surprisingly, it has been foundthat the epithelial lining extends sufficiently far into the orificeregion of the duct to permit successful labelling using generallyconventional immunocytochemical labelling reagents and techniques, asdescribed in more detail below. Exemplary tissue markers includeantigens and epitopes defined by the cytokeratins present in theepithelial cytoplasmic lining, such as cytokeratin 8, cytokeratin 18,and by molecules present in the membrane lining, such as E cadherin,epithelial membrane antigen (EMA), and the like. Suitable breastepithelial tissue markers are described, for example, in Moll et al.(1982) Cell 30:11-19; Gown and Vogel (1984) Am. J. Pathol. 114:309-321;and Johnson (1991) Cancer Metastasis Rev. 10:11-22.

[0025] Referring now to FIG. 3, an orifice region O of a ductal networkD is illustrated with a plurality of markers M lining the epithelium ofthe duct and extending to the perimeter of the orifice. Labelledantibodies A can be used to locate and label those markers M which arenear the orifice O. Frequently, it will be desirable or necessary towash the nipple with a solution capable of unblocking the orifice topermit binding of the antibodies or other labelling reagent. Forexample, the orifice can frequently become plugged withkeratin-containing materials, and washing with a keratinolytic solution,such as acetic acid (5% to 50% by weight) admixed in a pharmaceuticaldelivery vehicle, will expose sufficient marker sites at each orifice toenable labelling according to the methods of the present invention.

[0026] The labelled antibodies or other labelling reagents may beformulated as liquid, typically aqueous, solutions in a generallyconventional manner. Suitable anti-cytokeratin antibodies may beobtained from commercial suppliers, with specific antibodies includingFITC-anticytokeratin (Becton-Dickenson), CAM 5.2, and the like. Theantibodies may be coupled to one member of a signal-producing systemcapable of generating a detectable visual or other change on the tissuesurface, where an element will be referred to here and after as a“visual label.” Suitable signal-producing systems include fluorescentsystems, color-generating systems, and luminescent systems. Preferred isuse of fluorescent systems which comprise a single fluorescent label,but other systems which comprise two or more components includingenzymes, substrates, catalysts, enhancers, and the like, may also beemployed. At least one component of the signal-producing system will beattached to the antibody or other specific binding substance which iscapable of directly or indirectly binding to the tissue marker. Usually,the antibody will bind directly to the tissue marker, but it will alsobe possible to employ primary antibodies which are specific for thetissue marker and labelled secondary antibodies which introduce thelabel or component of the signal-producing system. For example, theprimary antibody can be mouse IgG and the labelled secondary antibodycan be FITC goat anti-mouse IgG (Zymed). Such signal-producing systemsand the use on tissue and tissue samples is well described in themedical, scientific, and patent literature relating toimmunocytochemical staining.

[0027] In an exemplary protocol according to the present invention, thenipple is first dekeratinized with 5% to 50% acetic acid to removekeratin and other potentially blocking and contaminating substances fromthe ductal orifices. A solution of the labelled antibody, preferably anantibody which directly binds to a cytokeratin or other epithelialcytoplasmic or surface membrane marker, such as the antibodies describedabove, is then applied to the nipple surface. The antibody is preferablylinked to a fluorescent marker, more preferably fluorescein, and thefluorescein-labelled antibody delivered in a buffered aqueous solution.Optionally, controls may be run. For example, labelled antibodies of thesame Ig class as the specific antibody may be exposed to the nipple atthe same dilution. By comparing the results with the specific antibodyand the control antibody, non-specific binding can be discounted.

[0028] The labelling reagent will typically be packaged (optionally withthe keratinolytic agent) together with instructions for use in aconventional assay package, such as a box, bottle, tube, pouch, or thelike. The instructions for use may be written out on a separateinstruction sheet or may be partially or fully incorporated onto thepackaging materials.

[0029] A second kit according to the present invention will comprise thelabelling reagent (optionally with the keratinolytic agent) in a packageas set forth above. The package will further include an access devicecapable of being introduced through the ductal orifice, such as acatheter, a fiber optic scope, or the like. Suitable catheters and fiberoptic scopes are described in the background art discussed above. Suchkits may further comprise instructions for use (IFU) setting forth anyof the methods described above.

[0030] The following experimental descriptions are offered by way ofillustration, not by way of imitation.

EXPERIMENTAL

[0031] A. Dekeratinizing the Nipple

[0032] Acetic acid is mixed with Velvacrol (50% v/w), a pharmaceuticalvehicle comprising an aqueous mixture of petrolatum/mineral oil, acetylalcohol, sodium laural sulfate, cholesterol, methylparaben,butylparaben, and propylparaben. To keep the acetic acid in solution,methyl cellulose (100 mg) is pre-added to the Velvacrol (5 g). Themixture possesses a uniform pasty consistency and is applied to thenipple as an ointment or paste. The keratinolytic agent is typicallyleft on the nipple for twenty-four hours or longer to remove the keratinplugs from the ductal orifices.

[0033] B. Labelling of the Ductal Orifice

[0034] For cytoplasmic antigens, the ductal epithelium must besolubilized with 70% by weight ethanol. For a membrane or surfaceantigen, the solubilization step is not necessary. A mouse monoclonalprimary antibody is used as a dilution of 1:5 to 1:100 and maintained onthe nipple for one hour at room temperature. After such incubation, thenipple is washed with phosphate buffered saline PBS and a secondaryantibody (fluoresceinated goat anti-mouse antibody) used at a dilutionof from 1:5 to 1:1000 fold at room temperature. After washing with PBS,the nipple may be examined under ultraviolet (UV) light at a wavelengthselected for the particular fluorochrome being used. A control can thenbe run using an antibody of a similar class, but without specificity forany of the ductal epithelial or other markers which may be present onthe nipple. This method will provide successful labelling of the ductalorifices and permit subsequent cannulation and examination of eachorifice.

[0035] C. Ductoscopy

[0036] Duct cannulation and exploration can be performed under white(visual) light. One or more ducts are cannulated first with a rigidmetal duct-probe (6 Fr Taber-Rothschild Galactography Kit, Manan MedicalProducts Inc., Northbrook, Ill.) dilated to 0.45 mm to 0.5 mm. A guidewire (0.4 mm) is then inserted, and a catheter passed over the guidewire. Physiological saline (0.2 ml to 0.5 ml) is instilled to wash theduct lumen. The washings may be spun down and analyzed cytologically.

[0037] The duct lumen is then dried by injecting 0.2 ml to 0.5 ml air.At the end of the final insufflation, the orifice is held shut bypinching the end of the nipple. An endoscope (FVS-3000, M&M Company,Tokyo), which is 0.4 mm in outer diameter is then threaded into the ductorifice while dilation of the duct with air is maintained. After theendoscope is inserted for 5 mm to 10 mm, its position may be confirmed.The cannulization may then be continued as far distally as possible.Desired diagnostic, therapeutic, or other material may then be instilledinto the duct.

[0038] Alternatively, after cannulation, the duct may be dilated withsaline using a closed system with a burst adapter to allow a betterview.

[0039] In a particular aspect of the present invention, cells may beremoved through the cannula (as washings). The collected cells may beprocessed according to standard cytological methods for similarwashings, such as bronchial washings and biopsy specimens. The cells maybe identified directly or indirectly by histopathological analysis ofthe duct from which the cells were obtained.

[0040] Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.

1. A method for obtaining fluid and cellular material from a breast ductcomprising: (a) locating at least one breast duct on a nipple of abreast, wherein the orifice of the breast duct is located by labelingcellular material at the orifice with a detectable label coupled to anantibody specific for a tissue marker characteristic of the tissue atthe ductal orifice and locating the orifice by observing the presence ofthe detectable label; (b) introducing washing fluid into the breast ductvia a catheter; and (c) collecting the washing fluid from the breastduct with the catheter, wherein the washing fluid collected comprisesfluid and cells from the breast duct.
 2. A method for obtaining fluidand cellular material from a breast duct comprising: (a) locating atleast one breast duct on a nipple of a breast, wherein the breast ductexhibits no observable spontaneous discharge; (b) introducing washingfluid into the breast duct via a catheter; and (c) collecting thewashing fluid from the breast duct with the catheter, wherein thewashing fluid collected comprises fluid and cells from the breast duct.3. A method according to claim 1, further comprising obtaining fluid andcellular material from multiple breast ducts.
 4. A method according toclaim 2, further comprising obtaining fluid and cellular material frommultiple breast ducts.
 5. A method according to claim 1 wherein thewashing fluid includes saline.
 6. A method according to claim 2 whereinthe washing fluid includes saline.
 7. A method according to claim 1,further comprising preparing the collected washing fluid for cytologicalanalysis.
 8. A method according to claim 2, further comprising preparingthe collected washing fluid for cytological analysis.
 9. A methodaccording to claim 7, wherein preparing the collected washing fluid forcytological analysis includes centrifugation.
 10. A method according toclaim 8, wherein preparing the collected washing fluid for cytologicalanalysis includes centrifugation.
 11. A method for obtaining fluid andcellular material from a breast duct for cytological analysiscomprising: (a) locating at least one breast duct on a nipple of abreast; (b) introducing washing fluid into the breast duct via acatheter; and (c) collecting the washing fluid from the breast duct withthe catheter, wherein the washing fluid collected comprises fluid andcells from the breast duct.
 12. A method according to claim 11, furthercomprising obtaining fluid and cellular material from multiple breastducts.
 13. A method according to claim 11 wherein the washing fluidincludes saline.
 14. A method according to claim 11, further comprisingpreparing the collected washing fluid for cytological analysis.
 15. Amethod according to claim 14, wherein preparing the collected washingfluid for cytological analysis includes centrifugation.